Antitumor properties of traditional lactic acid bacteria: Short-chain fatty acid production and interleukin 12 induction

This paper presents an in vitro evaluation of antitumor properties through producing short-chain fatty acids and inducing interleukin 12. In addition, it offers the most important and functional probiotic properties of 24 Lactobacillus gasseri, Lactiplantibacillus plantarum, Lactobacillus acidophilus, and Limosilactobacillus fermentum strains isolated from humans, foods, and fermented foods. To this end, survival in an acidic environment (pH = 2.5), tolerance in bile salt, viability in the presence of pepsin-pancreatin, adhesion percentage, antibiotic resistance, auto-aggregation, and potential percentage of co-aggregation are studied in contact with three human intestinal pathogens. These pathogens are Escherichia coli O157: H7 NCTC 12900, Salmonella enterica subsp. enterica ATCC 13076, and Listeria monocytogenes ATTC 7644. Also, in vitro induction amount of IL-12 in mouse splenocytes is investigated to evaluate antitumor properties by 19 strains of L. gasseri and L. plantarum along with the development of short-chain fatty acids (SCFA) by 5 strains of L. fermentum and L. acidophilus. Gas Chromatography Flame Ionization Detector (GC-FID) and enzyme-linked immunosorbent assay (ELISA) were used to measure short-chain fatty acids and IL-12, respectively. All strains had high viability under acidic conditions. The highest levels of pancreatin and pepsin resistance were found in strains LF56, LF57, LF55, OF, and F and strains LF56, LF57, and A7, respectively. All strains except LF56 had high resistance to bile salts. L. gasseri 54C had the highest average adhesion score (hydrophobicity) of 62.9 % among 19 strains. Despite the susceptibility of different strains of L. plantarum to the tested antibiotics, M8 and M11, S2G, A7, LF55, LF57, and 5G were resistant to kanamycin and chloramphenicol, respectively. Also, 21G was resistant to ampicillin, LF56 to tetracycline and M8, and M11, LF56, and 21G to Erythromycin. In addition, L. gasseri showed moderate resistance to ampicillin, erythromycin, and tetracycline, while L. fermentum ATCC 9338 showed good resistance to ampicillin, erythromycin, and chloramphenicol. In this respect, L. plantarum LF56 and gasseri 54C had the highest average auto-aggregation and co-aggregation against three pathogenic bacteria, respectively. The highest and lowest levels of acetic acid as short-chain fatty acids were produced by L. fermentum 19SH isolated from Horre 41.62 and L. fermentum 21SH from fermented seeds 27.047, respectively. Moreover, L. fermentum, with the OF code of traditional-fermented food origin, produced the most isobutyric acid, butyric acid, and valeric acid, with values of 0.6828, 0.74165, and 0.49915 mmol, respectively. L. fermentum isolated from the human origin with code F produced the most isovaleric acid of 1.1874 mmol. All the tested strains produced good propionic acid except L. fermentum 21SH from fermented seeds. Among strains, L. plantarum M11 isolated from milk and L. gasseri 52B from humans had the highest in vitro induction of IL-12, which is probably related to their cell wall compositions and structure.

isolated from milk and L. gasseri 52B from humans had the highest in vitro induction of IL-12, which is probably related to their cell wall compositions and structure.

Introduction
Probiotics are products containing an equivalent amount of living and advanced microorganisms that colonize and modify the microbiota of the host body, resulting in a beneficial effect [1].Regarding the positive effect of probiotics on the host body, they must be able to survive for a long time in the gastrointestinal tract.Probiotics are used to activate the immune system and inhibit certain infections, in addition to their use as growth stimulants [2].These compounds prevent cancer by influencing animal and human digestive enzymes, suppressing tumors and metabolites in vitro, and inhibiting carcinogens in vivo and in vitro [3].Probiotic bacteria have different functions depending on their origin.
In vitro, animal, human, and epidemiological experiments have shown that certain Lactobacillus strains can minimize the risk of different cancers by developing fermented products and preventing tumor development [4].Lactobacilli extracted from fermented milk products can enhance lactose digestion, regulate serum cholesterol levels, and control gastrointestinal diseases, certain forms of cancer in various medical and health areas (e.g., managing pregnancy infections), antibiotic-related diarrhea, and intestinal inflammation.In addition, they are effective in treating allergic diseases, preventing urinary tract infections, stimulating the immune system, and stabilizing the intestinal microbiota [5].Lactic acid bacteria isolated from yogurt also increase the immune system, reduce gastrointestinal infections, and lower cholesterol, cancer, and diarrhea.
Short-chain fatty acids are among the antitumor compounds developed by probiotic bacteria.These acids are classified as volatile fatty acids with 1-6 carbons that can be straight or branched and are absorbed in the large intestine.Acetic acid is the most concentrated short-chain fatty acid in the large intestine [6].Kahouli et al. [7] studied L. fermentum NCIMB 5221 for its ability to generate fatty acids and antitumor activity.These authors reported the antitumor production of the probiotic bacterium L. fermentum NCIMB 5221 as a producer of ferulic acid (FA) and an antioxidant and antitumor compound with the capacity to generate free fatty acids (FFAs).Wang et al. [8] investigated the synthesis of short-chain fatty acids by lactobacilli using Lactobacillus acidophilus RD758.According to this study, the fermentation temperature influences the fatty acid content of the L. acidophilus RD758 membrane, and the fermentation pH affects the concentration and form of fatty acid.
White blood cells and other cells secrete cytokines, as some low molecular weight proteins or glycoproteins, in response to certain stimulants.Interferon-gamma (IFN-γ) has shown an anti-proliferative and apoptotic induction effect on many receptor-induced tumor cells, indicating its defensive function against many tumors.Lactic acid bacteria, especially colon probiotic bacteria, promote health and modulate host immune responses [9].Tamang et al. [10] pointed out that synthesizing anti-inflammatory cytokines (e.g., IL-12 and IFN-γ) modulates T helper type 1/T helper type 2 (Th1/Th2) immune cells.
L. gasseri OLL2809 is a lactic acid bacterium that actively induces IL-12 (p70) [11].Also, the helper T cell is a type of T lymphocyte and white blood cell with a significant role in the body's immune system.Since IL-12 (p70) promotes the division of simple cells into Th1 cells, it can be used to assess Lactobacillus probiotics' anti-inflammatory effect [11].Research has shown that L. plantarum L137 and L. plantarum JCM1149 are active IL-12 (p70) inducers that can trigger Th1-type immune responses with anti-inflammatory and antitumor effects [12].The present study aims to screen four strains of traditional lactic acid bacteria (L.gasseri, L. plantarum, L. fermentum, and L. acidophilus) with antitumor properties in terms of producing short-chain fatty acids and inducing interleukin 12 and the relationship of these characteristics with probiotic properties.

Resistance of LAB strains to acidic conditions (low pH)
The strains were cultured for 48 h at 37 • C in MRS broth and centrifuged for 15 min at 6000×g.The supernatant was discarded, followed by washing the cells and suspending them in a phosphate buffer solution at pH = 2.5.After incubating at 37 • C for 4 h, the samples were diluted in sterile saline solution (0.85 % sodium chloride).Afterward, 100 μL of the last three dilutions were cultured on MRS agar, and the plates were incubated at 37 • C for 48 h.Finally, bacterial viability was measured using Eq. ( 1) [16]:

Resistance of LAB strains in bile salts
The MRS broth culture medium containing 0.3 % (w/v) of bile salts (bovine bile (Ibresco, Iran)) with 100 μL of grown bacteria was incubated for 4 h at 37 • C. Before and after incubation, 100 μL of successive dilutions was cultured on the surface of MRS agar and incubated for 48 h at 37 • C. The bacterial survival percentage was determined using Eq.(1) [17].

Simulation of gastric juice and LAB strains resistance
About 100 μL of grown bacteria was inoculated into the simulated gastric solution (3 mg/mL pepsin in 0.85 % sterile saline solution at pH = 2.5) and incubated at 37 • C for 4 h.Live cell count before and after incubation was determined by culturing 100 μL of successive dilutions on the surface of MRS agar medium and its incubation at 30 • C for 48 h [18].

Simulation of intestinal juice (pancreatin) and LAB strains resistance
Intestinal juice was prepared with 1 mg/mL pancreatin in 0.85 % saline solution (pH = 8).For this purpose, 100 μL of exponential phase bacteria was inoculated into pancreatin solution and kept at 37 • C for 6 h in an incubator.Afterward, live cell count before and after incubation was determined by preparing serial dilutions and culturing 100 μL of dilutions on MRS agar medium superficially.

Surface hydrophobicity of LAB strains
The exponential phase of bacteria was assessed by incubating the strains for 18-24 h at 30 • C in an MRS broth culture medium and then centrifuged at 5000×g for 10 min.The supernatant was discarded, and the cells were washed twice with 50 mM K 2 HPO 4 buffer at pH = 6.5.In the next step, the cells were suspended in phosphate-buffered saline (PBS; Pharmed, Iran) such that the cell density range was between 0.8 and 1 (spectrophotometer, lightwave S2000UV/Vis).About 3 mL of the microbial solution was mixed with 0.6 mL of n-Hexadecane into a test tube for 120 s, and the tubes were incubated at 37 • C for 30 min to separate into two phases.The upper phase was then cautiously separated, and the absorption of the lower phase was measured at a wavelength of 560 cm − 1 [19].
A 0 : Absorbance in the range of 0.8-1.
A: Lower phase absorption.

Antibiotic resistance
This experiment was carried out in 96-well cell culture plates using the broth microdilution method.First, 95 μL of MRS broth was added to each well.Except for the positive control row, 100 μL of ampicillin, kanamycin, erythromycin, chloramphenicol, and tetracycline antibiotics with an initial concentration of 1 mg/L were poured into the first well of each row, and then from the first to the second well, and so on.Dilution was completed up to the twelfth well.Next, 5 μL of each tested strain was added to each well, except to the negative control row, and the cell culture plates were kept at 37 • C for 24 h.The turbidity of the positive and negative control wells before warming and the rest after warming were read by ELISA reader (Stat fax 2100, USA).The lowest inhibitory concentration in the first well, lower than the positive control, was considered MIC.It is of note that positive and negative control wells do not contain antibiotics and bacteria, respectively [20].

Aggregative abilities of LAB strains
Auto-aggregation and co-aggregation of strains were investigated according to the method by Del et al. [21].Under the microaerophilic conditions, the strains were grown for 24 h at 37 • C in an MRS broth culture medium.After centrifugation (sigma 3-30K, Germany(at 1372×g for 10 min, the pellets were redissolved in 10 mL of PBS to around 10 8 CFU/mL (0.2 -0.3OD at 550 nm WL).The suspensions were mixed for 10 s and left at room temperature for 6 h without shaking.The absorbance was measured using a spectrophotometer at 600 nm while 1 mL of the mentioned suspension was collected every hour.The following formula was used to quantify the auto-aggregation percentage [20].
At: Absorption at different times.A0: Absorption at time zero.In this step, 3 mL of bacterial suspensions were subjected to experiments.Next, bacterial pathogens (i.e., E. coli O157: H7 NCTC 12900, S. enterica subsp.enterica ATCC 13076, and L. monocytogenes ATCC 7644) with a concentration of 10 8 CFU/mL and absorption (0.25-0.05OD at 600 nm WL) were mixed for 10 s and left at room temperature for 6 h without shaking to evaluate co-aggregation.Samples containing 6 mL of a bacterium suspension were used as a control.After a 6-h incubation at room temperature, 1 mL of this suspension was collected every hour, and the absorptions of the mixed suspension (probiotics and pathogens) and control were measured separately.The co-aggregation percentage was determined using Eq. ( 4), where AX and AY are the aggregations of bacterial strains and pathogens experimented with, respectively.Also, A (X + Y) denotes the total aggregation of bacterial strains and pathogens together [21].

Preparation of bacteria for producing short-chain fatty acids
Bacterial strains (L.fermentum with codes OF, F-ATCC9338, 19SH, 21SH, and L. acidophilus with code AC-ATCC4356) were transported to a fermentation container with 100 mL MRS broth medium and placed in an incubator at 37 • C. Next, they were shaken in the incubator at 4×g for 24 h to produce short-chain fatty acids, followed by transferring the culture medium to 50 mL of falcons.Because of creating the same conditions for all strains to produce a short-chain fatty acid, the same concentration (number of bacteria/ unit volume) with an absorbance of 0.8 at 660 nm equal to 3.6 × 10 6 CFU/mL of bacterial strains was considered in the study.After 24 h of fermentation, the medium was centrifuged for 10 min at 2800×g at a constant temperature of 4 • C to separate short-chain fatty released into the culture.The supernatant containing fatty acids was transferred to a 2-mL microtube and placed in a freezer at − 80 • C for 24 h [7].

Derivation of short-chain fatty acids
After thawing the frozen samples, they were centrifuged at 30 rpm for 30 min.The supernatant was then poured into a sterile microtube containing 300 μL of meta-phosphoric acid (Honeywell, Sweden) and vortexed.After that, the microtube was kept at 25 • C for 25 min and subsequently centrifuged at 2800g for 15 min.Next, the samples derived for analyzing short-chain fatty acids (SCFA) were injected into a GC-FID (Gas Chromatography-Flame Ionization Detector).The total contents of SCFA, propionate, acetate, and butyrate were calculated.Starting at 250 • C, the process was as follows: the sample (5 μl) was injected into the capillary column (Agilent HP-5 ms, the USA) at a split ratio of 25:1, which had been covered with a film, 0.15 μm in thickness, made from 80.2 % 1methylnaphthalene.The nitrogen flow rate in the mobile phase was first set at 1 mL/min and maintained at this value for 1 min.After that, it shifted to 0.8 mL/min for 1 min, then to 0.6 mL/min for 1 min, and finally back to 1 mL/min for 9.2 min.The FID temperature was adjusted at 260 • C. Next, the synthetic air and helium flow rates were changed to 30 and 350 ml/min, respectively.Lastly, the oven temperature was set at 100 • C, kept for 7 min, subsequently increased to 200 • C at a rate of 25 • C/min and maintained for 5 min.The concentrations of the SCFA were expressed as μmol/ml [22].

Induction of IL-12 (p70) by L. plantarum and L. gasseri strains
Five-week-old male BALB/c mice weighing 24 ± 1 gr were purchased from Iran's Pasteur Institute and kept on a regular diet.In all experiments, mice between the ages of 6 and 9 weeks were used.It is of note that Iran's Pasteur Institute has approved the experimental protocols used.Also, the code of ethics IR.UM.REC.1400.006 was received from the Biomedical Committee of the Ferdowsi University of Mashhad.

Preparation of heat-killed bacteria
The bacterial strains were incubated in MRS broth for 24 h at 37 • C and centrifuged at 4200×g for 4 min at 4 • C. Next, the pellet was washed with saline solution and distilled water three times.After making a pellet suspension in distilled water, 50 μL of bacteria suspension was inoculated into 5 mL of MRS broth culture medium (1 % V/V) with a (pH = 6.4) and incubated for 18 h at 37 • C. Afterward, the culture medium was added to 100 mL of MRS broth (Erlenmeyer flask) and shaken at 4×g at 37 • C for 18 h in a shaker incubator.The pH of the culture medium was regularly checked and kept at 6.4 or higher.The contents of the Erlenmeyer flask were placed into a pre-weighed falcon and centrifuged at 1000×g at 4 • C for 15 min.After discarding the supernatant, the pellet was washed twice with saline solution and once with distilled water (pellet weight was measured each time).Finally, the suspended pellet (2 mL in deionized water) was heated at 75 • C for 60 min in a hot water bath (MEMMERT WNE45).The killed cells were lyophilized and maintained to assay for producing IL-12 (p70) in vitro [11].

Statistical analysis
Data were expressed as the mean ± standard deviation.Each experiment was performed in triplicate.Statistical differences between or among the groups were analyzed using the one-way analysis of variance (ANOVA) with the Tukey mean comparison test.Differences were considered significant when the P-value was less than 0.05.The graphs were generated using GraphPad Prism software.

Acid tolerance of LAB strains
All strains of L. plantarum, acidophilus, fermentum, and gasseri showed tolerance to acidic conditions at a pH of 2.5.L. plantarum isolated from milk (i.e., M8, LF56, and LF57) and L. gasseri isolated from a healthy female vaginal (i.e., 54C) had the highest resistance to acid among these strains (Table 2).

Bile salt tolerance of LAB strains
Except for L. plantarum isolated from Lighvan cheese (LF56), almost all strains had a 100 % survival rate in the simulated bile salt of the pancreas (Table 3).

Tolerance to simulated intestinal juice (pancreatin)
L. fermentum isolated from camel Doogh (OF) and ATCC 9338 (F) and L. plantarum originated from Lighvan cheese (LF 56, LF 57, and LF 55) had the maximum survival rate in simulated intestinal juice (pancreatin) with a pH of 8.No colony was observed for L. plantarum isolated from milk (M8 and M11) and isolated from pitcher cheese (61G), L. gasseri with human source (49A), and L. acidophilus ATCC 4356 (AC) on the culture plate after 4 h of treatment (Table 5).

Surface hydrophobicity properties of LAB strains
In the present study, the strain with the highest degree of hydrophobicity was L. gasseri isolated from a human source, exhibiting a surface hydrophobicity of 56.56 %.Also, L. acidophilus ATCC 4356 (AC), L. plantarum isolated from the small intestine (A7), and L. plantarum isolated from fermented olive (21G) had moderate adhesions of 31.80,30.38, and 39.69 %, respectively.The majority of the strains had weak hydrophobicity (Fig. 1).

Antibiotic resistance test
Different strains of L. plantarum were sensitive to the tested antibiotics and resistant to kanamycin and chloramphenicol.Also, M11, S2G, A7, LF55, LF57, and 5G were resistant to chloramphenicol and M8, M11, and 61G to kanamycin.L. gasseri showed moderate resistance to ampicillin, erythromycin, and tetracycline, while L. fermentum ATCC 9338 (F) showed good resistance to ampicillin, erythromycin, and chloramphenicol.The highest antibiotic resistance for L. acidophilus ATCC 4356 (AC) is related to ampicillin, chloramphenicol, and tetracycline (Table 6).

Auto-aggregation and Co-aggregation abilities of LAB strains
In the auto-aggregation test, all strains had moderate sedimentation, except for L. gasseri (54C), isolated from human sources, which showed weak sedimentation.The highest auto-aggregation percentage was related to L. plantarum isolated from Ligvan cheese with code LF56 at the rate of 75 % (Fig. 2).Also, in the co-aggregation test with the pathogenic bacteria E. coli O157:H7 NCTC 12900, S. enterica subsp.enterica ATCC 13076, and L. monocytogenes ATTC 7644, the highest precipitation was related to the L. gasseri strain with code 54C on S. enterica subsp.enterica ATCC 13076 by 33.63 % (Fig. 3), E. coli by 31.93 % (Fig. 4), and L. monocytogenes by 24.98 % (Fig. 5).The sedimentation of the rest of the strains was almost at the same level [23].

Discussion
According to the FAO/WHO criteria for assessing probiotic properties, probiotic microorganisms should not only survive passage across the digestive tract but also proliferate in the intestine.This requirement guarantees their immunity to gastric juices and the ability to grow in the presence of bile under intestinal conditions or be consumed.Probiotic bacteria have antimicrobial activity against human pathogens and can bind to human mucosal cells [24].A study on probiotic properties reported that all strains of L. plantarum, acidophilus, fermentum, and gasseri have a strong tolerance to acidic conditions at a pH of 2.5.The strains of L. plantarum isolated from milk (M8) and (LF56 and LF57) from Lighvan cheese, and the strain of L. gasseri isolated from the vagina of a healthy woman (54C) had the best resistance to acid or low pH as their survival was 100 %.Acids in the human intestine (e.g., hydrochloric acid) are highly oxidizing and dissolve cell biomolecules such as fatty acids, proteins, and DNA molecules.Lowering the pH of the environment prevents metabolism and decreases the growth and survival of lactic acid bacteria [25].Tolerance to the acidic conditions of these bacteria is related to their inherent properties.The explanation is that L. plantarum is a heterofermentative bacterium that can ferment raffinose polysaccharides and other carbohydrates, leading to its higher acid tolerance than other lactobacilli.Also, L. gasseri is widely found in the human intestine and gastrointestinal tract, especially in breast milk, and can easily withstand the stomach's acidic environment [26][27][28].
In addition, the majority of 19 strains had 100 % viability in the synthetic bile salt of pancreatic juice, except for L. plantarum isolated from Lighvan cheese (LF56).In this respect, there is a comparatively high quantity of bile salt in the small and large intestines, which is poisonous and harmful to living organisms.Therefore, resistance and stability in the presence of bile salts are among the most important characteristics of probiotic bacteria to consider when choosing them [29].As bacteria are exposed to bile salts, their cellular homeostasis is disrupted, and the degradation of lipid membranes and cell membrane proteins results in bacterial material damage and cell death [30].Some strains' resistance to bile salts is related to bile salt hydrolysis activity, as bile salt hydrolysis lowers toxicity and side effects of bile salt [31].
In the present study, the survival rates of strains A7, LF56, and LF57 in simulated gastric juice were 100 %, indicating the strain- specific ability to withstand the acidic conditions of the gastric environment.This result highlights the importance of strain selection in determining the efficacy of probiotic survival in the gastrointestinal tract.Previous research has also emphasized the significance of strain-specific characteristics in probiotic functionality and survival in the human digestive system [32].Even certain foods can help environmental bacteria survive in the human body.However, these factors are not considered in our simulated model of the gastrointestinal tract.According to the literature, Lactobacillus tolerance to gastric juice depends on the activity of the ATPaz hydrogen pump and the composition of bacterial membranes.Furthermore, it depends on the bacteria, the culture medium, and the incubation conditions [33].

AC
Another excellent aspect of probiotic bacteria is their ability to colonize the intestinal wall.As a result, accessibility to the intestinal epithelium (also known as a precondition for colonization) is a critical indicator and requirement when selecting probiotic bacteria [34].L. gasseri can be isolated from intestinal tissue, oral cavity, vaginal region, urine, and human blood, suggesting the strong bond of this bacterium with the human body.Regarding the difference in the amount of D-alanine and the proportion of hexose group in the cell wall liposuction anchor relative to other sources, this strain has the highest ability to bind to intestinal epithelial cells [35].The results of the colonization of bacteria (especially L. gasseri) in this study are consistent with those of previous studies.
Co-aggregation is a highly efficient probiotic process that inhibits pathogens from adhering to the surface of intestinal epithelial cells [36].The ability of the probiotic bacteria to interact with pathogens and competition with them for adhesion to the intestinal epithelial cell surface has been significantly proven.This dependence is due to the presence of molecules on the membrane surface of probiotic strains of lactic acid bacteria that act as ligands to bind to the pathogens.Consistent with the findings of the previous studies, the human strain of L. gasseri had the highest adhesion and cohesive properties [20].
Several strains of L. plantarum were sensitive to the studied antibiotic substance.However, M8 and 21G were resistant to kanamycin and ampicillin, respectively, and M11, S2G, A7, LF55, 5G, and LF57 showed high resistance against chloramphenicol.In addition, L. gasseri had medium resistance to ampicillin, erythromycin, and tetracycline, while L. fermentum showed high resistance to chloramphenicol and ampicillin.Some probiotic bacteria resist various antibiotics such as vancomycin, streptomycin, gentamicin, and ciprofloxacin.The explanation is that the factors effective in antibiotic resistance (e.g., antibiotic-resistant genes) can be transferred from one lactobacillus to another and, most importantly, to pathogenic bacteria such as staphylococcus [37].
Prior to applying these probiotic bacteria, more research is required to comprehensively assess the profiles of their antibiotic resistance as well as their ability to transfer resistance genes to pathogens.Further studies should also be conducted on the screening of the genotypic and phenotypic characteristics of antibiotic resistance, particularly against classes concentrate critical antibiotics such as aminoglycosides, cephalosporins, and quinolones, while lacking transferable resistance genes such as parC, aac(6′) Ii, ermB, ermC, and tetM.It is necessary to understand and mitigate the antibiotic resistance of probiotic bacteria to ensure that they are effective and safe for use in food and pharmaceutical applications, guaranteeing comprehensive investigation prior to extensive implementation [38].
Joghatai et al. [39] examined the probiotic properties of L. plantarum, fermentum, and acidophilus isolated from fermented and human sources.In this research, the L. fermentum isolated from Horre (19SH) had the highest survival rate in the simulated environment with gastric acid (96 %) and co-aggregation with S. enterica subsp.entericaserovar typhimurium (51 %).Moreover, L. fermentum (OF) isolated from traditionally fermented foods was among the strains with the highest survival in acidic environments.
Since the highest amount of fatty acid produced was from L. fermentum, they might have differences in the cell wall.Cell wall thickness increases during short-chain fatty acid production in the logarithmic phase, which varies among strains.In this research, 5 strains of probiotic bacteria from human, fermentative, and dairy sources produced short-chain fatty acids such as acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid.L. fermentum (19 SH) isolated from Horre (traditionalfermented food) produced the highest amount of acetic acid as short-chain fatty acids.Besides, the maximum quantities of propionic, isobutyric, butyric, and valeric acid were produced by L. fermentum (OF) isolated from camel doogh (traditional-fermented food).L. fermentum (ATCC 9338) (F) generated the maximum level of producing isovaleric acid from a human source.The highest volume of fatty acids released by L. fermentum was due to a difference in the cell wall between L. fermentum and acidophilus.Tanigawa and Umezu [40] compared the cell wall thickness of L. fermentum H-34, L. heterohiochii H-1, Leuconostoc mesenteroides (IFO3832), and Latilactobacillus sakei (IFO3541) isolated from fermented foods.The results revealed that L. fermentum (H-34) and L. heterohiochii (H-1) had thicker cell walls than the control bacteria (i.e., 600 Å versus 200 Å for the control bacteria).In addition, findings showed that wall thickness increases with increasing bacterial growth as a function of the bacterial growth period [40].A similar study investigated fatty acids formed by L. fermentum (NCIMB 5221) and L. acidophilus (RD758) isolated from fermented foods.The results showed that L. fermentum (NCIMB 5221) was able to produce ferulic acid (FA) as an antioxidant and antitumor compound.Moreover, they produced free fatty acids (FFA), thereby inhibiting the growth of colon cancer cells [7,8].Acetic acid is the most prevalent short-chain fatty acid in the large intestine [6].In this research, most of the acetic acid was produced by L. fermentum (19SH).Due to the large intestine's high amount of acetic acid, more than half of all short-chain fatty acids in the feces are acetic acid [41].The acetic acid concentration in the large intestine exceeds that of all short-chain fatty acids in cells, making it a key component in carbohydrate and fat metabolism [42].One of the most important effects of short-chain fatty acids is that they lower pH, thereby lowering the population of pathogenic microorganisms and improving nutrient absorption.In addition, a compound like a butyrate can modify intestinal epithelial cells and increase mucus production, which helps bacteria adhere better [43].Research has shown that L. fermentum (NCIMB 5221) prevents the development of colon cancer cells by producing short-chain fatty acids while promoting the growth of intestinal epithelial cells.Lactobacillus proliferation is highly effective in the proliferation of cancer cells, thereby damaging cancer cells by preventing their proliferation.Besides, they are beneficial to normal colon cells.This effect is strongly linked to these bacteria' ability to produce more short-chain fatty acids, especially acetic, butyric, and propionic acid, compared to other probiotic bacteria [7].Among SCFAs, butyric acid has the highest anti-inflammatory activity.The energy loss originates from the intestinal mucosa's inflammatory phase, which appears in combination with multiple pathological processes.In this regard, butyric acid is the primary energy source for intestinal epithelial cells, wherein butyric acid has a beneficial immune system regulating effect on intestinal epithelial cells and other mucosal cells.Butyrate induces apoptosis in colorectal cancer cells and lymphomas [44,45].Butyrate also inhibits the growth and development of colon stem cells, which are found at the end of the intestine.Several human studies have shown significantly lower butyrate levels in patients with colorectal cancer than in healthy people [46].In the present study, the L. fermentum strain from camel doogh produced the most butyric acid (0.74 mmol).In contrast to other short-chain fatty acids, the role of valeric acid in the gut is unclear.Valeric acid has been shown in a few experiments to promote the development of intestinal epithelium and to be active in diseases including colitis, heart-metabolic diseases, and cancer [47,48].The strain of L. fermentum (ATCC 9338) (F) from a human produced the most isovaleric acid (1.18 mmol) in this study.Propionic acid is primarily formed by Gram-positive bacteria in the human intestine [49].In the liver, this acid inhibits gluconeogenesis and cholesterol synthesis [50].It also has antibacterial, anti-inflammatory, and antitumor properties, thereby protecting the human gut from microbial pathogens [51,52].In this study, L. fermentum (OF) isolated from camel doogh provided the most propionic acid (1.43 mmol).In previous research, prescriptions for lactic acid bacteria and fermented dairy goods have been found to affect the activities of digestive enzymes related to intestinal cancer compounds.Several intestinal bacteria can transform non-toxic compounds into metabolites that cause tumorigenesis and inflammation [53].Since probiotic bacteria contain lactic and acetic acids, they lower intestinal pH and establish an atmosphere conducive to removing intestinal microbial pathogens and modifying bacterial enzymes [54].In this respect, previous studies have shown that administering probiotic bacteria such as L. acidophilus to patients with colon cancer reduced fecal pH, decreased infectious bacteria, and increased antitumor and anticancer effects significantly [55].
According to the ELISA results, the highest induction of IL-12 in the culture of lymphocyte cells was related to L. plantarum (8SH) isolated from Sauerkraut, a traditionally fermented food, L. plantarum (M11) isolated from milk, and L. gasseri (52B) isolated from the vagina.On the other hand, the lowest amount is related to L. plantarum (10SH) isolated from Tarkhineh (12.5),L. plantarum (LF57) isolated from Lighvan cheese (14.16), and L. gasseri (49A) isolated from the vagina (12.16).More adhesion to the intestine's inner wall or more colonization in the intestine is among the important factors for inducing IL-12 in mouse splenocytes by bacteria.This factor can be estimated using amounts of adhesion, auto-aggregation, and co-aggregation of bacteria.
According to Joghataei et al. [39], L. plantarum (8SH) isolated from Sauerkraut (due to more tendency to adhesion or colonization greatly) decreases the adhesion of Escherichia coli bacteria to HT-29 cells.Also, this bacterium inhibited the development of E. coli O157: H7 and S. enterica subsp.entericaserovar typhimurium (ATCC 14028).The hydrophobicity level of this bacterium was 33 %, while its auto-aggregation and co-aggregation with E. coli and S. enterica subsp.entericaserovar typhimurium was reported as 51 %, 31 %, and 41 %, respectively.According to this study, the hydrophobicity levels of L. plantarum (M11) and L. gasseri (52B) were 25 % and 57 %, respectively.Also, the result of auto-aggregation was 46 % and 40 %, respectively, and the rate of co-aggregation on the three pathogens studied was about 5 %.The present study showed that L. gasseri (52B) has the highest adhesion level, with the same amount of auto-aggregation.Meanwhile, L. plantarm (8SH) had the highest co-aggregation.The ability of both probiotic and lactic acid strains to interact with pathogens and compete for adherence to the intestinal epithelial cell surface was consistent with those reported in similar studies.Since the aggressive potential of the pathogenic bacteria seems to be related to the ability to bind to epithelial cells, the impact of auto-aggregation and co-aggregation is very significant for probiotic bacteria.This factor can be estimated using amounts of adhesion, auto-aggregation, and co-aggregation of bacteria.This dependence can be due to the presence of certain molecules on the membrane surface of probiotic lactic acid bacteria strains that function as ligands and bind to pathogens or in the position of antibiotic resistance.Lactic acid bacteria strains are bound to the intestinal epithelial cells by a bandage [20].However, among these three strains, L. plantarum (M11) had the highest rate of IL-12 induction, which is typically related to its cell wall.Murasaki et al. [12] investigated the stimulation of IL-12 by L. plantarum L-137 cell wall lipoteichoic acid compared to L. plantarum JCM1149.According to their results, the lipoteichoic acid genes synthesized from L. plantarum L-137's cell wall induced more IL-12 (p40) in mouse splenocytes and dendritic cells than in the control group.Cell wall components in gram-positive bacteria (e.g., lipoteichoic acid and non-methyl P. Mobasherpour et al. peptidoglycans) also stimulate immune cells.According to these researchers, the compound had no additive effect on the induction of IL-12 (p40) in their experiments on peptidoglycans.TLR2 receptors initiate signals in response to molecular patterns associated with pathogens and inflammatory agents.These receptors are stimulated by L. plantarum L137 lipoteichoic acid, but peptidoglycans are not altered [12].Kaji et al. [56] compared lipoteichoic acid in Lacticaseibacillus casei, L.plantarum, and L.gasseri bacteria in terms of IL-10 and IL-12 induction.According to these authors, in lactic acid bacteria, the teichoic acid of the cell wall is spread from peptidoglycan and lipoteichoic acid from the plasma membrane.It has been determined that the lipoteichoic acid of L. plantarum has an incremental effect on IL-12 induction.Grangette et al. [57] investigated the immune system and the induction of inflammatory cytokines such as IL-10 and IL-12 by L. plantarum NCIMB8826 cell wall lipoteichoic acid.These researchers reported that cell wall mutations that affect lipoteichoic acid D-alanylation (reduction of D-alanine lipoteichoic acid and glucose replacement) result in a sharp increase in IL-10 induction [57].The cell wall of probiotic bacteria plays a significant role in how well they adhere to free intestinal toxins.According to previous studies, the structure of probiotic bacteria is associated with a decrease in tumorigenesis [58].Lactobacilli cellular components (e.g., entire cells, heat-inactivated cells, cell walls, peptidoglycans, and cytoplasmic extracts) have various functions when exposed to cancer cells [59].Reiki et al. [60] found that Lactobacillus cytoplasmic extract and cell wall decreased the protein level of the P53 mutant gene significantly.The P53 gene is a tumor suppressor gene that plays an important role in cell apoptosis.This gene acts as a guard for the cell and inhibits the replication process in the damaged DNA of the cell.Probiotic bacteria, like lactobacilli, inhibit many pathological cell types.This type of bacteria strengthens the host's immune system and helps prevent cancer by inhibiting the expression of malignant tumors [31].Increased levels of cytokines and immunoglobulins, increased mononucleate cell proliferation, activation of natural macrophages, and inhibitory macrophages are all effects of probiotic bacteria on the immune system.The stimulation of immunity against pathogenic bacteria and protozoa is referred to as autoimmunity.T cells' cytokine production and lymphocyte differentiation are also suppressed by probiotic bacteria.All bacteria have been shown to stimulate immune cell proliferation and the synthesis of pro-inflammatory cytokines, including TNF-α and IL-12 [61,62].Most significantly, probiotic bacteria have these beneficial effects on the immune system without causing an inflammatory response that is harmful to the body.The immune response can be enhanced when many probiotic bacteria are taken together and function synergistically.This result is most common when Lactobacillus and Bifidobacteria are consumed together.

Conclusion
Lactic acid bacteria strains with a high potential to adhere to intestinal epithelial cells colonize the intestine more effectively than other strains.These strains function as pathogen inhibition using various mechanisms, including antimicrobial activity, co-aggregation with pathogens, and attachment to intestinal epithelial cells.L. gasseri strain isolated from the vagina (54C) is the strongest probiotic strain among the lactic acid bacteria examined in this study.They have a significant level of antimicrobial activity and a strong capacity to prevent pathogen infection through co-aggregation.Different variations of lactic acid bacterial strains may also inhibit pathogen invasion, confirming the importance of creating new and specialized probiotic compounds to prevent infections and the host's health.IL-12 plays an essential role in controlling the function of probiotic compounds, improving the response of Th1 cells, and regulating cellular immune activity in particular.Therefore, the potential of a lactobacillus strain to induce IL-12 induction may be a useful measure of immune stimulatory operation.The composition of lactobacilli's cell walls is a key component in inducing IL-12 release and effectively stimulates macrophages and dendritic cells to metabolize IL-12.L. plantarum isolated from Sauerkraut (8SH), L. plantarum isolated from milk (M11), and L. gasseri isolated from the vagina (52B) showed the maximum induction of IL-12.Also, it is also predicted that L. plantarum isolated from milk and L. gasseri from the vagina has beneficial immunological properties due to the presence of strong adhesion genes.Factors such as microbial growth curve and choosing the right extraction method are effective in producing short-chain fatty acids as antitumor compounds.L. fermentum (19SH) and (OF) isolated from traditional fermented foods are a viable alternative owing to their high development of antitumor compounds, including acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid.This effect is mostly attributed to its thicker cell wall than L. acidophilus (ATCC 4356) (AC).The ability of probiotic bacteria strains to survive in the gastrointestinal tract is an exciting and significant feature, most probably regarding their cell wall strength and stiffness.

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Fig. 5 .
Fig. 5. Comparison of co-aggregation abilities percentages among different LAB strains and Listeria monocytogenes.

Table 1
Characteristics of lactic acid bacteria strains used in this study.

Table 2
Acid tolerance of LAB strains in PBS (PH = 2.5).

Table 3
Bile salt tolerance of LAB strains.

Table 6
Minimum inhibitory concentration (MIC, mg/l) of 5 antibiotics on LAB strains.